Sunflower regeneration through embryogenesis and organogenesis

ABSTRACT

The present invention relates to the regeneration of sunflowers via embryogenesis and organogenesis. The process comprises the steps of: 
     (a) culturing tissue obtained from a sunflower plant on a first medium which comprises mineral salts, vitamins, amino acids, sucrose and a hormone in an amount sufficient to ensure callus formation; 
     (b) optionally subculturing said callus on a maintenance medium which comprises mineral salts, vitamins, amino acids, sucrose and a hormone in an amount sufficient for callus maintenance; 
     (c) subculturing said callus on a second medium which comprises mineral salts, vitamins, amino acids, sucrose and a hormone in an amount sufficient to precondition the callus; 
     (d) subculturing said callus on a third medium which comprises mineral salts, vitamins, sucrose and a hormone in an amount sufficient to ensure shoot formation, and 
     (e) subculturing said shoot on a fourth medium which comprises mineral salts, vitamins, sucrose and a hormone in an amount sufficient to ensure root formation, whereby plants are obtained.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for the regeneration ofsunflower plants from cell or tissue culture through embryogenesis andorganogenesis. More specifically, cells or tissues of sunflower plantsare cultured to produce calli. The calli can then be cultured for aprolonged period. The calli are then cultured on a preconditioningmedium. The preconditioned calli are cultured to produce shoots whichare cultured to produce roots whereby sunflower plants are produced. Thepresent invention also relates to the sunflower plants and seeds whichare produced by this method.

2. Description of the Prior Art

Several methods have been described in the prior art which result in theregeneration of sunflower. All of these methods have involved the use oforganogenesis. However, these methods do not appear to be veryefficient, and only result in the formation of a few regenerated plants.In organogenesis, plant parts are cultured on a first medium to inducecallus formation. The callus can then be transferred to a second mediumto induce shoot formation. The shoots are then transferred to a thirdmedium to induce root formation, at which point the regeneratedplantlets (plants) can be transferred to soil.

One example of inefficient organogenesis can be found in Sadu, Indian J.Exp. Biol., pages 110-111 (June 1974). Sadu reported the regeneration ofsunflower by culturing stem pith tissue to form a callus whichdifferentiated to form plantlets. The medium was the same throughout theregeneration scheme.

The medium used to form the callus and to differentiate into plants wasa modified White's medium containing 1 ppm of the hormone indole aceticacid (IAA). The modified White's medium contained the followingingredients in one liter of medium:

    ______________________________________                                        Component Weight (mg) Component  Weight (mg)                                  ______________________________________                                        NH.sub.4 NO.sub.3                                                                       400         ZnSO.sub.4 2.7                                          KCl       65          thiamine   0.2                                          KNO.sub.3 80          nicotinic acid                                                                           0.5                                          KH.sub.2 PO.sub.4                                                                       12.5        pyridoxine 0.5                                          Ca(NO.sub.3).sub.2.H.sub.2 O                                                            144         glycine    2.0                                          MgSO.sub.4.7H.sub.2 O                                                                   7.2         inositol   100                                          Na.sub.2 EDTA                                                                           25          sucrose    20000                                        H.sub.3 BO.sub.3                                                                        1.6         agar       6000                                         MnSO.sub.4.4H.sub.2 O                                                                   6.5                                                                 ______________________________________                                    

It was found that 2,4-dichlorophenoxyacetic acid (2,4-D) promoted callusgrowth but did not support differentiation of the callus. The additionof 0.1 ppm kinetin resulted in callus growth but no differentiation.

Chandler and Jan describe the regneration of sunflower from immatureembryos of interspecific crosses. The immature embryos are grown ongrowth medium as described by Chandler and Beard, below. After 2 to 3weeks, the enlarged embryos are transferred to tubes containing MSmedium (MS mineral salts, vitamins and sucrose) containing IAA andkinetin, generally used at 0.3 ppm and 1.0 ppm, respectively. The tubesare cultured at 28° C. in the light to produce calli which then formshoots. Shoots are usually transferred to a MS medium containing nohormones but containing 0.2% activated charcoal to cause root formation.The plantlets are then transferred to soil.

Heaton discloses the regeneration of the domestic sunflower Helianthusannus L. from immature embryos. The immature embryos were plated onGibco Murashige Shoot-Tip Rooting Medium. Callus formation was inducedusing 3% sucrose, 0.3 mg/l IAA and 1.0 mg/l kinetin. The embryos werekept in the dark for two weeks, then in the light for 12 hours per day.After five weeks, shoots formed on the callus. The shoots weretransferred to rooting medium for plantlet production.

Binding et al, Z. Pflanazenphysiol. 101, 119 (1981) describe theregeneration of shoots of the sunflower Helianthus annus fromprotoplasts. Protoplasts were cultured in V-KM medium to form callus,and the calli were transferred to a low osmotic medium. The low osmoticmedium comprises B5 medium with 15 μM 6-benzylaminopurine (BA). Shootsformed on this medium.

Rogers et al, In Vitro 9, 463 (1974) describe the production of rootsfrom a callus in Helianthus annus. This was performed by culturing planttissue sequentially on three media. The first medium comprises MS basalmedium with 1 mg/l α-naphthalene acetic acid (NAA), 1 mg/l kinetin and 5mg/l 2,4-D. The second medium comprises MS basal medium with 0.5 mg/lkinetin and 2 mg/l IAA. The third medium comprises 0.05 mg./l kinetinand 0.1 mg/l IAA. No whole plants were produced by this method.

One additional method has been described to produce sunflower plantsfrom an embryo. This method has found use in producing plants fromembryos which do not develop in the original plant itself, eitherbecause of embryo abortion or seed dormancy resulting from thehybridization of non-compatible species. This method is embryo culture,and has been described by Chandler and Beard in The Sunflower, pages45-47 (August/September 1980) and Crop Science 23, 1004 (1983). Thisprocess involves the rescue of the embryo prior to abortion followed bymaturation of the embryo and germination to form the plant.

In this process, young embryos were isolated 3 to 7 days afterpollination. These embryos were usually less than 0.1 mm in diameter.The embryos were plated on a solid, growth medium which contained B5salts, vitamins, amino acids, the auxin α-naphthalene acetic acid (NAA)at a concentration of 0.05 mg/l, and 12% sucrose. It was found that if9% sucrose was utilized instead of 12% and 1.0 mg/l indoleacetic acid(IAA) was used instead of NAA, then the young embryos has a tendency togrow as undifferentiated callus instead of embryos. In addition, veryyoung embryos also germinated prematurely on this latter medium.

After 1 to 2 weeks, the enlarged (2-6 mm in diameter) embryos weretransferred to a liquid medium for germination of the embryos to plants.During the enlargement period, some of the embryos began root formationand pigment synthesis. The embryos were matured to the cotyledon stagein order to obtain plant formation. The liquid, germination mediumcontained B5 salts and 1% sucrose. After the embryo generated roots anda shoot, it was transplanted to soil.

The present invention is the first instance of a general method forobtaining sunflower plants, i.e., regenerating sunflowers, through theuse of embryogenesis and organogenesis. Sunflower plants and seeds areproduced by this process. The sunflower plants resulting from thisprocess may differ from the starting plant material as a result ofsomoclonal variation. The pathway is also useful in that it will enablethe use of various selection processes to provide further variation. Theplants which are produced can then be used in conventional breedingprograms.

SUMMARY OF THE INVENTION

The present invention is directed to a process for regeneratingsunflowers, particularly to regenerating cultivars of domesticsunflower, Helianthus annus. This process uses embryogenesis andorganogenesis. The process comprises the steps of inducing callusformation on an induction medium from tissue of a sunflower plant,maintaining the calli, culturing the calli on a preconditioning medium,forming shoots on a shoot forming medium and forming roots on a rootforming medium.

More specifically, the present process comprises the steps of:

(a) culturing tissue obtained from a sunflower plant on a first mediumwhich comprises mineral salts, vitamins, amino acids, sucrose and ahormone in an amount sufficient to ensure embryogenic callus formation;

(b) optionally subculturing said callus on a maintenance medium whichcomprises mineral salts, vitamins, amino acids, sucrose and a hormone inan amount sufficient for embryogenic callus maintenance,

(c) subculturing the calli on a second medium which comprises mineralsalts, vitamins, amino acids, sucrose and a hormone in an amountsufficient to precondition said callus.

(d) subculturing said calli on a third medium which comprises mineralsalts, vitamins, sucrose and a hormone in an amount sufficient to ensureshoot formation, and

(e) subculturing said shoot on a fourth medium which comprises mineralsalts, vitamins, sucrose, and a hormone in an amount sufficient toensure root formation, whereby plants are obtained.

The source of tissue is preferably immature embryos from the HA 300, RHA271, and HA 89 cultivars of Helianthus annus. The first, maintenance andsecond media preferably contain modified B5 mineral salts, the vitaminsand amino acids as described by Chandler and Beard, supra. The thirdmedium preferably contains MS mineral salts and vitamins, described byHenderson et al, Am.J.Botany 39, 467 (1952) which have been modified.The fourth medium preferably contains MS mineral salts.

The preferred hormones are 2,4-D and abscisic acid (ABA) in the firstmedium, ABA in the maintenance medium, BA or ABA and BA in the secondmedium, IAA and kinetin in the third medium, and IAA in the fourthmedium.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a process for regeneratingsunflowers, especially the domestic sunflower Helianthus annus, viaembryogenesis and organogenesis. In this process, embryogenic callus isformed which is preconditioned so that shoots are first obtained fromtissue culture and then roots. The plants are then placed in soil forgrowth to maturation. The present invention is also directed tosunflower plants obtained by this process and to seeds from theseplants.

In general, the process comprises (a) culturing tissue obtained from asunflower plant on a first medium to produce embryogenic calli, (b)optionally culturing the calli on a maintenance medium to maintain theembryogenic calli, (c) culturing the calli on a second medium toprecondition the calli, (d) culturing the preconditioned calli on athird medium to produce shoots, and (e) culturing the shoots on a fourthmedium to produce roots. After plantlets have been developed, they canbe grown in soil.

The plant tissue which is preferred for use in the initiation of callusis the immature embryo. The immature embryos with pericarps are isolatedfrom the sunflower heads when the embryos are in the range of 0.5 to 2.0mm. The embryos are sterilized with bleach and rinsed with sterilewater. The immature embryos are isolated from the pericarps and platedonto an embryogenic callus induction medium, hereinafter referred to asthe first medium.

The medium comprises mineral salts, vitamins, amino acids, sucrose and ahormone in an amount sufficient for callus formation. The mineral saltscomprise macroelements and microelements. The macroelements used in thefirst medium may be the following compounds: magnesium sulfate, calciumchloride, monosodium phosphate, potassium nitrate and ammonium sulfate.The microelements contained in this medium are: boric acid, manganesesulfate, zinc sulfate, sodium molybdate (VI), copper (II) sulfate,cobalt chloride, potassium iodide, iron (II) sulfate and disodiumethylenediaminetetraacetic acid (EDTA). This combination of mineralsalts is known in the art as the B5 mineral salts. In the first medium,the B5 mineral salts have been modified so that the medium contains lessiron and EDTA than the standard B5 mineral salts. It is also preferredto use less copper and iodine.

In this medium, the iron is chelated by the EDTA. Citric acid can beutilized in place of the EDTA as the chelating agent. In a preferredembodiment, chelated iron is added in preparing the medium rather thanadding iron (II) sulfate and disodium-EDTA.

The preferred amounts of the macroelements and microelements which areused to prepare one liter of medium are as follows: 250 mg magnesiumsulfate heptahydrate, 150 mg calcium chloride dihydrate, 150 mgmonosodium phosphate monohydrate, 2500 mg potassium nitrate, 134 mgammonium sulfate, 3 mg boric acid, 10 mg manganese sulfate monohydrate,2 mg zinc sulfate heptahydrate, 0.25 mg sodium molybdate (VI) dihydrate,0.0025 mg copper (II) sulfate pentahydrate, 0.025 mg cobalt chloridehexahydrate, 0.075 mg potassium iodide, 2.78 mg iron (II) sulfateheptahydrate, and 3.73 mg disodium-EDTA.

In a more preferred embodiment, the iron concentration is 0.20-3.25mg/l, preferrably 0.81 mg/l. This may be added to the medium in any ofthe forms described above.

The first medium also contains vitamins. The vitamins which are utilizedinclude nicotinic acid, thiamine, pyridoxine and myo-inositol. Thevitamins have been described by Chandler and Beard, supra. The preferredamounts of vitamins needed to prepare one liter of medium are: 1 mgnicotinic acid, 10 mg thiamine hydrochloride, 1 mg pyridoxinehydrochloride and 4000 mg myo-inositol. This combination of vitaminswill be referred to as the Chandler vitamins.

The first medium further contains amino acids. The amino acids are:alanine, glutamine, serine, tryptophan and cysteine. All amino acids arein the L-form unless otherwise indicated. The amino acids have beendescribed by Chandler and Beard, supra. The preferred amounts of aminoacids used to prepare one liter of medium are: 1000 mg alanine, 800 mgglutamine, 160 mg serine, 50 mg tryptophan, and 10 mg cysteine. Thiscombination of amino acids will be referred to as the Chandler aminoacids.

The first medium also contains sucrose and a hormone. The sucrose isutilized in the amount of 7%-14%, with 12% being preferred. The hormonewhich may be utilized is a mixture of ABA and 2,4-D, 1.0-7.0 μM ABA and5.0-12.0 μM 2,4-D are utilized. Preferably, 5 μM ABA and 10 μM 2,4-D areused. Agar is used to solidify the medium. A final concentration of 0.7%has been found to be satisfactory. The medium has a pH of 5.5-6.0, witha preferred pH of 5.8.

The medium is sterilized by autoclaving all components except thevitamins and amino acids which are sterilized via microporous membranefiltration.

The immature embryos are plated on the first medium and cultured in thedark for about 7 to 14 days. It is preferred to use 7 to 10 days ofculturing. During this time, the embryo undergoes dedifferentiation andcallus formation.

After culturing the embryos on the first medium, the callus which isformed may optionally be transfered and subcultured on a maintenancemedium before transfer to the second medium. When this step isperformed, the callus is subcultured on this medium for about 2 weeks inthe dark. The callus can be subcultured on this medium for a prolongedperiod with transfer occurring at approximately 2 week intervals.

The maintenance medium comprises mineral salts, vitamins, amino acids,sucrose and a hormone in an amount sufficient to maintain embryogeniccallus. The mineral salts comprise macroelements and microelements. Themacroelements and microelements are as described for the first medium.The vitamins and amino acids are as described for the first medium. Thesucrose concentration is 7%-14%, with 12% being preferred. The hormoneis preferably ABA, and is used in an amount of 0.05-1.0 μM. It ispreferred to use 1 μM ABA. Agar is added to the medium to solidify it. Aconcentration of 0.7% is satisfactory. The medium has a pH of 5.5-6.0,with 5.8 preferred. The medium is sterilized as described above.

After culturing the callus on the maintenance medium for 2 weeks, ordesired intervals thereof, the callus is transferred and subcultured ona preconditioning medium, hereinafter referred to as the second medium.The callus is subcultured on this medium for 1 to 2 weeks, preferably 1week, in the dark. If the callus from the first medium is not culturedon the maintenance medium, it is transferred directly to the secondmedium.

The second medium comprises mineral salts, vitamins, amino acids,sucrose and a hormone in an amount to precondition the callus. Themineral salts, vitamins and amino acids are as described for the firstmedium. The sucrose concentration is 4%-8%, with 6% being preferred. Thehormone is preferably BA or a mixture of ABA and BA in an amount of0.5-2.0 μM BA or a mixture of 1.0-4.0 μM ABA and 0.5-2.0 μM BA. It ispreferred to use 3 μM ABA and 1 μM BA. Agar is added to the medium tosolidify it. 0.7% has been found to be satisfactory. The medium has a pHof 5.5-6.0, with 5.8 preferred. The medium is sterilized as describedabove.

After culturing the callus on the second medium, the callus istransferred and subcultured on a shoot formation medium, hereinafterreferred to as the third medium. The callus is subcultured on the thirdmedium for 2 to 4 weeks in the light, with a photoperiod of 12 to 16hours per day, preferably 16 hours per day. During this time, shoots areformed on the callus.

The third medium comprises mineral salts, vitamins, sucrose and IAA inan amount sufficient for shoot formation. The mineral salts comprisemacroelements and microelements. The macroelements which are utilized inthe fourth medium are: magnesium sulfate, calcium chloride,monopotassium phosphate, potassium nitrate and ammonium nitrate. Themicroelements contained in this medium are: boric acid, manganesesulfate, zinc sulfate, sodium molybdate (VI), copper (II) sulfate,cobalt chloride, potassium iodide, iron (II) sulfate and disodium-EDTA.This combination of mineral salts is known in the art as the MS mineralsalts.

The preferred amounts of the macroelements and microelements which areused to prepare one liter of medium are: 370 mg magnesium sulfateheptahydrate, 440 mg calcium chloride dihydrate, 170 mg monopotassiumphosphate, 1900 mg potassium nitrate, 1650 mg ammonium nitrate, 6.2 mgboric acid, 16.9 mg manganese sulfate monohydrate, 8.6 mg zinc sulfateheptahydrate, 0.25 mg sodium molybdate (VI) dihydrate, 0.025 mg copper(II) sulfate pentahydrate, 0.025 mg cobalt chloride hexahydrate, 0.83 mgpotassium iodide, 27.8 mg iron (II) sulfate heptahydrate, and 37.3 mgdisodium-EDTA.

The third medium further contains vitamins. The vitamins which arepresent in this medium include thiamine, pyridoxine, myo-inositol,riboflavin, pantothenate, p-aminobenzoic acid, niacin, choline, folicacid and biotin. These vitamins have been described by Henderson et al,supra. The preferred amounts of the vitamins used to prepare one literof medium are: 0.5 mg thiamine hydrochloride, 0.1 mg pyridoxinehydrochloride, 100.5 mg myo-inositol, 0.05 mg riboflavin, 0.8 mg calciumpantothenate, 0.05 mg p-aminobenzoic acid, 0.5 mg niacin, 0.1 mg cholinehydrochloride, 0.1 mg folic acid, and 0.005 mg biotin. This combinationof vitamins will be referred to as the Henderson vitamins which havebeen modified to contain 100 mg/l more myo-inositol and 0.4 mg/l morethiamine hydrochloride.

The third medium also contains sucrose and a hormone, which ispreferably a mixture of IAA and kinetin. The sucrose is utilized in theamount of 2%-4%, with 3% being preferred. The IAA is utilized in anamount of 0.3-3.0 μM IAA and 0.5-9.0 μM kinetin. It is preferred to use0.3 μM IAA and 5 μM kinetin. Agar is added to the medium to solidify it.A concentration of 0.7% is satisfactory for this purpose. The medium hasa pH 5.5-6.0, with 5.8 preferred. The medium is sterilized as describedabove.

The shoots are transferred from the third medium to the fourth medium,when they are 2 cm long. If the shoots have not elongated properly or ifthe shoots begin in vitro flowering, they may be transferred to freshthird medium or to the third medium having either (a) IAA in an amountdescribed above and reduced kinetin (generally, 0.5-4.5 μM with 2.0 μMpreferred), or (b) 15-25 μM adenine sulfate and containing no IAA. Theshoots are subcultured on a solid, root formation medium, hereinafterreferred to as the fourth medium. The shoots are subcultured on thismedium for 6 to 10 days in the light, with a photoperiod of 12-16 hoursper day, preferably 16 hours per day.

The fourth medium comprises mineral salts, vitamins, sucrose and ahormone. The mineral salts comprise macroelements and microelements. Themacroelements and microelements are as described for the third medium.The vitamins are myo-inositol and thiamine. Preferably, these arepresent in the amounts of 100 mg/l of myo-inositol and 0.4 mg/l ofthiamine hydrochloride. The sucrose concentration is 2%-3%, with 2%being preferred. Agar is added to solidify the medium. A concentrationof 0.7% is satisfactory for this purpose. The hormone is preferably IAAand is used in an amount of 0.05-0.5 μM, with 0.1 μM being preferred.The fourth medium is sterilized by autoclaving, and has a pH of 5.5-6.0,with a preferred pH of 5.8.

After 5 to 14 days on the fourth medium, the plantlets can betransferred to soil and the greenhouse. This is generally accomplishedby transferring the plantlets to soil which is well moistened andcontained in a high humidity chamber. Once the plantlets areestablished, they are removed from the high humidity chamber,transplanted to soil, and grown to maturity. Seeds are produced by themature plants.

The above process is useful for regenerating plantlets from tissue ofmany cultivars of domestic sunflower. The process is especially usefulfor regenerating plantlets from Helianthus annus cv. HA 89, HA 300 andRHA 271.

The present invention will be further described by reference to thefollowing non-limiting examples. When the materials are cultured in thelight, it is understood to mean light having a photoperiod of 16 hoursper day and at a temperature of 25°-29° C. unless indicated otherwise.

EXAMPLE 1 Preparation of Stock Solutions 1. Mineral Salts

A. Modified B5

A 10X modified B5 mineral salts stock solution was prepared bydissolving the following ingredients in 1000 ml of distilled, deionizedwater.

    ______________________________________                                        Component Weight (mg) Component   Weight (mg)                                 ______________________________________                                        MgSO.sub.4.7H.sub.2 O                                                                   2500        ZnSO.sub.4.7H.sub.2 O                                                                     20                                          CaCl.sub.2.2H.sub.2 O                                                                   1500        Na.sub.2 MoO.sub.4.2H.sub.2 O                                                             2.5                                         NaH.sub.2 PO.sub.4.H.sub.2 O                                                            1500        CuSO.sub.4.5H.sub.2 O                                                                     0.025                                       KNO.sub.3 25000       CoCl.sub.2.6H.sub.2 O                                                                     0.25                                        (NH.sub.4).sub.2 SO.sub.4                                                               1340        KI          0.75                                        H.sub.3 BO.sub.3                                                                        30          FeSO.sub.4.7H.sub.2 O                                                                     27.8                                        MnSO.sub.4.H.sub.2 O                                                                    100         Na.sub.2 EDTA                                                                             37.3                                        ______________________________________                                    

The stock solution was divided into 100 ml aliquots.

2. Vitamins and Amino Acids

A. Chandler Vitamins and Amino Acids

A 40X stock solution of Chandler vitamins and amino acids was prepared.The stock solution was made by dissolving the following components in500 ml of distilled, deionized water.

    ______________________________________                                        Component Weight (g)   Component Weight (g)                                   ______________________________________                                        nicotinic acid                                                                          0.02         glutamine 16                                           thiamine.HCl                                                                            0.2          serine    3.2                                          pyridoxine.HCl                                                                          0.02         tryptophan                                                                              1.0                                          myo-inositol                                                                            80           cysteine  0.2                                          alanine   20                                                                  ______________________________________                                    

This solution was sterilized by membrane filtration using an 0.2μ Gelmanfilter prior to addition to the media.

B. Henderson Vitamins

A 1000X stock solution of Henderson vitamins (unmodified) was preparedby dissolving the following components in 200 ml of distilled, deionizedwater.

    ______________________________________                                        Component Weight (mg) Component  Weight (mg)                                  ______________________________________                                        thiamine.HCl                                                                            20          p-amino-   10                                                                 benzoic acid                                            pyridoxine.HCl                                                                          20          niacin     100                                          myo-inositol                                                                            100         choline.HCl                                                                              20                                           riboflavin                                                                              10          folic acid 20                                           Ca pantothenate                                                                         160         biotin     1                                            ______________________________________                                    

This solution was sterilized by membrane filtration prior to use asdescribed above.

3. Hormones

A. A 1 mM stock solution of 2,4-D was prepared by dissolving 0.1105 g of2,4-D in a couple ml of 1M KOH and diluting to 500 ml with distilled,deionized water.

B. A 2 mM stock solution of IAA was prepared by dissolving 0.0876 g ofIAA in a couple ml of 1M KOH and diluting to 250 ml with distilled,deionized water.

C. A 1 mM stock solution of kinetin was prepared by dissolving 0.108 gof kinetin in a couple ml of 1M HCl and diluting to 500 ml withdistilled, deionized water.

D. A 1 mM stock solution of ABA was prepared by dissolving 0.132 g ofABA in a couple ml of 1M KOH and diluting to 500 ml with distilled,deionized water.

E. A 1 mM stock solution of BA was prepared by dissolving 0.113 g of BAin a couple ml of 1M HCl and diluting to 500 ml with distilled,deionized water.

F. A 1 mg/ml stock solution of adenine sulfate was prepared bydissolving 100 mg of adenine sulfate in 100 ml of distilled, deionizedwater.

EXAMPLE 2

A. First Medium or Callus Induction Medium

The first medium was prepared by adding 10 ml of the 2,4-D stocksolution, 5 ml of the ABA stock solution, 120 g of sucrose and 7 g ofagar to 100 ml of the modified B5 stock solution, and the volume broughtto 975 ml with distilled, deionized water. The pH was adjusted to 5.8with 1M KOH and the mixture autoclaved at 120 psi for 15 minutes. 25 mlof the Chandler vitamins and amino acids stock solution, which had beensterilized as described above, was added to the cooling medium which wasthen poured into petri dishes.

To prepare first medium with different concentrations of 2,4-D and ABA,the appropriate amount of the 2,4-D and ABA stock solutions were used.For example, to prepare a first medium having 5 μM 2,4-D and 1 μM ABAinstead of 10 μM 2,4-D, and 5 μM ABA 5 ml of the 2,4-D stock solutionand 1 ml of the ABA stock solution were used.

B. Callus Maintenance Medium

The maintenance medium was prepared by adding 1 ml of the ABA stocksolution, 120 g of sucrose and 7 g of agar to 100 ml of the modified B5stock solution, and the volume brought to 975 ml with distilled,deionized water. The pH was adjusted to 5.8 with 1M KOH and the mixtureautoclaved at 120 psi for 15 minutes. 25 ml of the Chandler vitamins andamino acids stock solution, sterilized by membrane filtration asdescribed above, was added to the cooling medium which was then pouredinto petri dishes.

To prepare maintenance medium having a different concentration of ABA,the appropriate amount of the ABA stock solution was used in the mannerdescribed above.

C. Second Medium or Callus Preconditioning Medium

The second medium was prepared by adding 3 ml of the ABA stock solution,1 ml of the BA stock solution, 60 g of sucrose and 7 g of agar to 100 mlof the modified B5 stock solution, and the volume brough to 975 ml withdistilled, deionized water. The pH was adjusted to 5.8 with 1M KOH andthe mixture autoclaved at 120 psi for 15 minutes. 25 ml of the Chandlervitamins and amino acids stock solution, sterilized by the membranefiltration as described above, was added to the cooling medium which wasthen poured into petri dishes.

To prepare second medium having different concentrations of ABA and BA,the appropriate amounts of the ABA and BA stock solutions were used inthe manner as described above.

D. Third Medium or Shoot Formation Medium

The third medium was prepared by dissolving one packet of powderedMurashige minimal organics medium without sucrose (obtained from GibcoLaboratories and which contains the MS mineral salts, 100 mgmyo-inositol and 0.4 mg thiamine hydrochloride), 30 g of sucrose and 7 gof agar in 500 ml of distilled, deionized water. 0.05 ml of the IAAstock solution and 5 ml of the kinetin stock solution were then added,and the volume brought to 999 ml with distilled, deionized water. The pHwas adjusted to 5.8 with 1M KOH and the mixture autoclaved at 120 psifor 15 minutes. 1 ml of the Henderson vitamins stock solution,sterilized as described above, was added to the cooling medium. Themixture was then poured into petri dishes.

The procedure described above was utilized to prepare third mediumhaving different IAA and kinetin concentrations.

To prepare the third medium containing adenine sulfate instead of IAAand kinetin, the desired amount of the adenine sulfate stock solutionwas added in place of the IAA and kinetin stock solutions. For example,to prepare this medium having 22 μM adenine sulfate, 4 ml of the adeninesulfate stock solution was added.

E. Fourth Medium or Root Formation Medium

The fourth medium was prepared by dissolving one packet of powderedMurashige minimal organics medium without sucrose, 20 g of sucrose and 7g of agar in 1000 ml of distilled, deionized water. 0.05 ml of the IAAstock solution was then added. The pH was adjusted to 5.8 with 1M KOH.The mixture was autoclaved at 120 psi for 15 minutes. The cooling mediumwas poured into petri dishes.

To prepare the fourth medium having a different IAA concentration, theappropriate amount of the stock solution was added as described above.

EXAMPLE 3

Sunflower Regeneration

Immature embryos with pericarps were isolated from the head of thesunflower Helianthus annus cv. RHA 271 when they were 0.5 to 2 mm insize. RHA 271 was obtained from Sigco Research, Incorporated. The emryoswith pericarps were sterilized with a 20% bleach solution for 10minutes. They were then rinsed with sterile water. The immature embryoswere separated from the pericarps, endosperm and embryo sac and platedonto the first medium, contained in a petri dish. The first medium wasprepared as described in the preceding example, using 10 μM 2,4-D and 5μM ABA. The petri dish was placed in the dark and cultured for 10 daysto form calli.

At this time, each callus was transferred to the maintenance medium,which was prepared as described above, using 1 μM ABA, and alsocontained in a petri dish. The callus was cultured on this medium for 10days in the dark.

Each callus was then transferred to the second medium. The third mediumwas prepared as described in Example 2, using 3 μM ABA and 1 μM BA. Thecallus was cultured on this medium for 10 days in the dark.

At this time, each callus was transferred to the third medium, which wasprepared as described above, using 0.3 μM IAA and 5 μM kinetin, and alsocontained in a petri dish. The callus was cultured on this medium for 14days in the light. The callus differentiated to form shoots.

The shoots 2 cm long were then transferred to the fourth medium, alsocontained in petri dishes. The fourth medium was prepared as describedabove, using 0.1 μM IAA. The shoots were cultured on this medium for 9days in the light. During this time period, the shoots formed roots. Theshoots which were not 2 cm long or which had begun in vitro floweringwere transferred to fresh third medium containing 0.3 μM IAA and 2 μMkinetin, and cultured in the light for 8 days before being transferredto the fourth medium.

After 9 days, the plantlets were transferred to soil in the greenhouse.The plantlets were planted in cubes, and the soil was well moistened.The cubes were placed in a sweater box and covered with a second sweaterbox to maintain a high humidity environment. The soil was kept wellmoistened for 5 days, after which the plantlets were transferred to 12inch pots. The pots were watered three times weekly and fertilized every10 days. The plants were hand-pollinated, and maintained until the seedswere mature. Seeds were then harvested and stored for future use. Someseeds from one of the regenerants were planted and germinated to producesunflower plants.

EXAMPLES 4-18

The above procedure was essentially followed with some variation in theculture periods using different cultivars and different hromoneconcentrations. Helianthus annus cv. HA 89 and HA 300 were obtained fromSigco Research, Incorporated.

    __________________________________________________________________________    HORMONE CONCENTRATION (μM)                                                           First Medium                                                                         Maint. Medium                                                                         Second Medium                                                                         Third Medium                                                                          Fourth Medium                        Example                                                                            Cultivar                                                                           2,4-D                                                                             ABA                                                                              ABA     ABA BA  IAA Kinetin                                                                           IAA                                  __________________________________________________________________________    4    RHA 271                                                                            12  1  0.5     4   0.5 3.0 0.5 0.5                                  5    HA 89                                                                              10  4  1.0     3   1.5 1   7.0 0.1                                  6    HA 89                                                                              5   7  0.75    4   0.5 0.3 5   0.05                                 7    RHA 271                                                                            8   8  0.05    1   2   0.3 9   0.05                                 8    HA 89                                                                              10  2  0.5     3   1   0.5 9   0.5                                  9    HA 89                                                                              10  5  --      2   2   2   4   0.1                                  10   HA 300                                                                             5   7  1.0     2   1   0.3 5   0.1                                  11   HA 300                                                                             10  4  0.5     3   1   3   0.5 0.3                                  12   RHA 271                                                                            5   7  1       3   1   2   2   0.4                                  13   HA 89                                                                              10  5  0.1     3   0.5 1   3   0.1                                  14   HA 89                                                                              12  1  1.0     2   1.5 0.3 5   0.2                                  15   HA 300                                                                             10  5  1.0     1   2   2   7   0.1                                  16   RHA 271                                                                            10  5  --      2   2   0.3 5   0.3                                  17   HA 89                                                                              5   5  1.0     3   1   0.3 5   0.5                                  18   RHA 271                                                                            10  4  1.0     --  1   0.5 6   0.1                                  __________________________________________________________________________

Some seeds from some of the HA 89 regenerants were planted andgerminated to produce sunflower plants.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known and customary practice withinthe art to which the invention pertains.

What is claimed is:
 1. A process for regenerating sunflower plants fromcell or tissue culture through embryogenesis and organogenesis whichcomprises the steps of:(a) culturing tissue obtained from a sunflowerplant on a first medium which comprises mineral salts, vitamins, aminoacids, sucrose and a mixture of abscisic acid (ABA) and2,4-dichlorophenoxyacetic acid (2,4-D) in an amount sufficient to ensurecallus formation; (b) subculturing said callus on a second medium whichcomprises mineral salts, vitamins, amino acids, sucrose and6-benzylaminopurine (BA) or a mixture of ABA and BA in an amountsufficient to ensure callus preconditioning; (c) subculturing saidpreconditioned callus on a third medium which comprises mineral salts,vitamins, sucrose and a mixture of indoleacetic acid (IAA) and kinetinin an amount sufficient to ensure shoot formation, and (d) subculturingsaid shoot on a fourth medium which comprises mineral salts, vitamins,sucrose and IAA in an amount sufficient to ensure root formation,whereby plants are obtained.
 2. The process of claim 1 wherein saidtissue is immature embryos.
 3. The process of claim 1 wherein saidcallus is subcultured on a maintenance medium which comprises mineralsalts, vitamins, amino acids, sucrose and ABA in an amount sufficient toensure callus maintenance prior to subculturing on said second medium.4. The process of claim 3 wherein the concentrations of ABA, 2,4-D, IAA,BA and kinetin are:(1) 5.0-12.0 μM 2,4-D and 1.0-7.0 μM ABA in saidfirst medium; (2) 0.05-1.0 μM ABA in said maintenance medium; (3)0.5-2.0 μM BA or 1.0-4.0 μM ABA and 0.5-2.0 μM BA in said second medium;(4) 0.3-3.0 μM IAA and 0.5-9.0 μM kinetin in said third medium, and (5)0.05-0.5 μM IAA in said fourth medium.
 5. The process of claim 4 whereinthe concentration of sucrose is:(1) 7%-14% in said first medium; (2)7%-14% in said maintenance medium; (3) 4%-8% in said second medium; (4)2%-4% in said third medium, and (5) 2%-3% in said fourth medium.
 6. Theprocess of claim 5 wherein said mineral salts of said first, maintenanceand second media comprise magnesium sulfate, calcium chloride,monosodium phosphate, potassium nitrate, ammonium sulfate, boric acid,manganese sulfate, zinc sulfate, sodium molybdate, copper sulfate,cobalt chloride, potassium iodide and chelated iron.
 7. The process ofclaim 6 wherein said mineral salts are the B5 mineral salts modified sothat the concentration of the copper sulfate is 0.0025 mg/l and theconcentration of the potassium iodide is 0.075 mg/l and the chelatediron contains 0.81 mg/l iron.
 8. The process of claim 5 wherein saidvitamins of said first, maintenance and second media comprise nicotinicacid, thiamine, pyridoxine and myo-inositol.
 9. The process of claim 8wherein said vitamins are the Chandler vitamins.
 10. The process ofclaim 5 wherein said amino acids of said first, maintenance and secondmedia are alanine, glutamine, serine, tryptophan and cysteine.
 11. Theprocess of claim 10 wherein said amino acids are the Chandler aminoacids.
 12. The process of claim 5 wherein said mineral salts of saidthird and fourth medium comprise magnesium sulfate, calcium chloride,monopotassium phosphate, potassium nitrate, ammonium nitrate, boricacid, manganese sulfate, zinc sulfate, sodium molybdate, copper sulfate,cobalt chloride, potassium iodide, iron sulfate and disodium-EDTA. 13.The process of claim 12 wherein said mineral salts are the MS mineralsalts.
 14. The process of claim 5 wherein said vitamins of said thirdmedium are thiamine, pyridoxine, myo-inositol, riboflavin, calciumpantothenate, p-aminobenzoic acid, niacin, choline and folic acid. 15.The process of claim 14 wherein said vitamins are the Hendersonvitamins, modified to contain 100.5 mg/l myo-inositol and 0.5 mg/lthiamine hydrochloride.
 16. The process of claim 5 wherein said vitaminsof said fourth medium are myo-inositol and thiamine.
 17. A process forregenerating sunflower plants from cell or tissue culture throughembryogenesis and organogenesis which comprises the steps of:(a)culturing tissue obtained from a sunflower plant on a first medium whichcomprises mineral salts consisting of magnesium sulfate, calciumchloride, monosodium phosphate, potassium nitrate, ammonium sulfate,boric acid, manganese sulfate, zinc sulfate, sodium molybdate, coppersulfate, cobalt chloride, potassium iodide and chelated iron, vitaminsconsisting of nicotinic acid, thiamine, pyridoxine and myo-inositol,amino acids consisting of alanine, glutamine, serine, tryptophan andcysteine, sucrose and a mixture of 2,4-dichloropehnoxyacetic acid(2,4-D) and abscisic acid (ABA) in an amount sufficient to ensure callusformation; (b) subculturing said callus on a second medium whichcomprises mineral salts consisting of magnesium sulfate, calciumchloride, monosodium phosphate, potassium nitrate, ammonium sulfate,boric acid, manganese sulfate, zinc sulfate, sodium molybdate, coppersulfate, cobalt chloride, potassium iodide and chelated iron, vitaminsconsisting of nicotinic acid, thiamine, pyridoxine and myo-inositol,amino acids consisting of alanine, glutamine, serine, tryptophan andcysteine, sucrose and 6-benzylaminopurine (BA) or a mixture of ABA andBA in an amount sufficient to ensure callus preconditioning; (c)subculturing said preconditioned callus on a third medium whichcomprises mineral salts consisting of magnesium sulfate, calciumchloride, monopotassium phosphate, potassium nitrate, ammonium nitrate,boric acid, manganese sulfate, zinc sulfate, sodium molybdate, coppersulfate, cobalt chloride, potassium iodide, iron sulfate anddisodium-EDTA, vitamins consisting of thiamine, pyridoxine,myo-inositol, riboflavin, calcium pantothenate, p-aminobenzoic acid,niacin, choline and folic acid, sucrose and a mixture of indoleaceticacid (IAA) and kinetin in an amount sufficient to ensure shootformation, and (d) subculturing said shoot on a fourth medium whichcomprises mineral salts consisting of magnesium sulfate, calciumchloride, monopotassium phosphate, potassium nitrate, ammonium nitrate,boric acid, manganese sulfate, zinc sulfate, sodium molybdate, coppersulfate, cobalt chloride, potassium iodide, iron sulfate anddisodium-EDTA, vitamins consisting of myo-inositol and thiamine, sucroseand IAA in an amount sufficient to ensure root formation, whereby plantsare obtained.
 18. The process of claim 17 wherein said tissue isimmature embryos.
 19. The process of claim 17 wherein said callus issubcultured on a maintenance medium which comprises mineral saltsconsisting of magnesium sulfate, calcium chloride, monosodium phosphate,potassium nitrate, ammonium sulfate, boric acid, manganese sulfate, zincsulfate, sodium molybdate, copper sulfate, cobalt chloride, potassiumiodide and chelated iron, vitamins consisting of nicotinic acid,thiamine, pyridoxine and myo-inositol, amino acids consisting ofalanine, glutamine, serine, tryptophan and cysteine, sucrose and ABA inan amount sufficient to ensure callus maintenance prior to subculturingon said second medium.
 20. The process of claim 19 wherein theconcentrations of ABA, 2,4-D, IAA, BA and kinetin are:(1) 5.0-12.0 μM2,4-D and 1.0-7.0 μM ABA in said first medium; (2) 0.05-1.0 μM ABA insaid second medium; (3) 0.5-2.0 μM BA or 1.0-4.0 μM ABA and 0.5-2.0 μMBA in said second medium; (4) 0.3-3.0 μM IAA and 0.5-9.0 μM kinetin insaid third medium, and (5) 0.05-0.5 μM IAA in said fourth medium. 21.The process of claim 20 wherein the concentration of sucrose is:(1)7%-14% in said first medium; (2) 7%-14% in said maintenance medium; (3)6%-8% in said second medium; (4) 2%-4% in said third medium, and (5)2%-3% in said fourth medium.
 22. The process of claim 21 wherein saidmineral salts of said first, maintenance and second media are the B5mineral salts modified so that the concentration of the copper sulfateis 0.0025 mg/l and the concentration of the potassium iodide is 0.075mg/l and the chelated iron contains 0.81 mg/l iron.
 23. The process ofclaim 21 wherein said vitamins of said first, maintenance and secondmedia are Chandler vitamins.
 24. The process of claim 21 wherein saidamino acids of said first, maintenance and second media are Chandleramino acids.
 25. The process of claim 21 wherein said mineral salts ofsaid third and fourth media are MS mineral salts.
 26. The process ofclaim 21 wherein said vitamins of said third medium are Hendersonvitamins, modified to contain 100.5 mg/l myo-inositol and 0.5 mg/lthiamine hydrochloride.
 27. The process of claim 21 wherein theconcentration is said fourth medium of myo-inositol is 100 mg/l and ofthiamine hydrochloride is 0.4 mg/l.
 28. A process for regeneratingsunflower plants from cell or tissue culture through embryogenesis andorganogenesis which comprises the steps of:(a) culturing tissue obtainedfrom a sunflower plant on a first medium which comprises the B5 mineralsalts modified so that the concentration of the copper sulfate is 0.0025mg/l and the concentration of the potassium iodide is 0.075 mg/l and thechelated iron contains 0.81 mg/l iron, the Chandler vitamins, theChandler amino acids, 7%-14% sucrose and a mixture of 1.0-7.0 μMabscisic acid (ABA) and 5.0-12.0 μM 2,4-dichlorophenoxyacetic acid(2,4-D) to ensure callus formation; (b) subculturing said callus on asecond medium which comprises B5 mineral salts modified so that theconcentration of the copper sulfate is 0.0025 mg/l and the concentrationof the potassium iodide is 0.075 mg/l and the chelated iron contains0.81 mg/l iron, the Chandler vitamins, the Chandler amino acids, 4%-8%sucrose and 0.5-2.0 μM 6-benzylaminopurine (BA) or a mixture of 1.0-4.0μM ABA and 0.5-2.0 μM BA to ensure callus preconditioning; (c)subculturing said preconditioned callus on a third medium whichcomprises the MS mineral salts, the Henderson vitamins modified tocontain 100.5 mg/l myo-inositol and 0.5 mg/l thiamine hydrochloride,2%-4% sucrose and a mixture of 0.3-3.0 μM indoleacetic acid (IAA) and0.5-9.0 μM kinetin, to ensure shoot formation, and (d) subculturing saidshoot on a fourth medium which comprises the MS mineral salts, 100 mg/lmyo-inositol, 0.4 mg/l thiamine hydrochloride, 2%-3% sucrose, and0.05-0.5 μM IAA to ensure root formation, whereby plants are obtained.29. The process of claim 28 wherein said tissue is immature embryos. 30.The process of claim 28 wherein said callus is subcultured on amaintenance medium which comprises B5 mineral salts modified so that theconcentration of the copper sulfate is 0.0025 mg/l and the concentrationof the potassium iodide is 0.075 mg/l and the chelated iron contains0.81 mg/l iron, the Chandler vitamins, the Chandler amino acids, 7%-14%sucrose and 0.05-1.0 μM ABA to ensure callus maintenance prior tosubculturing on said second medium.
 31. The process of claim 30 whereinthe concentration of sucrose is:(1) 12% in said first medium; (2) 12% insaid maintenance medium; (3) 6% in said second medium; (4) 3% in saidthird medium, and (5) 2% in said fourth medium.
 32. The process of claim31 wherein the concentrations of ABA, 2,4-D, BA, IAA and kinetin are:(1)10 μM 2,4-D and 5 μM ABA in said first medium; (2) 1 μM ABA in saidmaintenance medium; (3) 1 μM BA or 3 μM ABA and 1 μM BA in said secondmedium; (4) 0.3 μM IAA and 5.0 μM kinetin in said third medium, and (5)0.1 μM IAA in said fourth medium.